The overall goal of this experiment is to determine NK cell count and cytotoxic activation levels in a human whole blood sample in a fast, reliable and stable manner. This method can help answer questions in the immunological fields, such as, What are the effects of outside interventions on the bioactivity of NK cells? The main advantage of this technique is that it is fast and accurate, While still providing reproducible results across experiments.
To isolate NK cells from human whole blood, first place 15 milliliter tubes labeled Whole blood sample, Negative Fraction and Positive Fraction into the appropriate positions in the cell separator tube rack. When all of the tubes are loaded, insert the separator rack onto the mini-sampler for auto labeling, and select Reagent on the menu, to highlight the position where the vial will be placed on the rack. Select Read Reagent to activate the 2D code reader and hold the appropriate reagent vial in front of the 2D code reader at an angle 0.5 to 2.5 centimeters from the code reader cover.
Place the read vial into the appropriate separator rack position. Next, under the Separation tab, highlight the desired positions. Then open the Labeling submenu and select an auto-labeling program.
Assign the cell separation reagent microbeads to rack positions 1, 2, 3 and 4. And select the Posselwb separaton program. Select the Rinse Wash program.
Enter 1500 microliters for the Sample Volume under the Volume sub menu, and press Enter. Then click Run to begin the cell separation. Clicking okay to confirm that enough buffer is available for processing all of the samples.
To prepare a Cytotoxicity Assay sample, first, label 1.5 milliliter tubes for each sample and/or participant as appropriate, and pipe out the desired ratio of NK cells and DIO labeled K562 cells into each tube. Collect the cells by centrifugation, and carefully remove the supernatant without disturbing the cell pellet. Next, resuspend the cell mixture in 500 microliters of incomplete NK cell medium, and label the cells in each tube with 5 microliters of propidium iodide.
Collect the cells via another centrifugation, and incubate the cull culture at 37 degrees celsius for two hours. At the end of the incubation, centrifuge the cells under the same centrifuge conditions, and resuspend the pellet in 25 microliters of fresh incomplete NK cell culture medium. To analyze the cells by Flow Cytometry, first, click the start up button in the imaging flow cytometer software to flush the system.
Next, click on the scatterplot button to create four scatterplots, and load the double positive control tube. Using the double positive control sample, determine the desired intensity for the 405 milliwatt laser, so that the detector is not overloaded, and set the imager to acquire the appropriate number of cell counts. Setting the 405 milliwatt laser at an intensity that is suitable for the detection of both DIO and PI will require experience with both dyes and the detection range of the flow cytometer.
After the acquisition of the double positive control sample finishes, load the DIO only control sample, select the 40X objective, and turn off the 642 milliwatt laser and the Brightfield channel. Breed the appropriate number of cells and repeat the acquisition for the propidium iodide only sample. When all of the control samples have been run, turn the 405 milliwatt laser and Brightfield channel, back on, and click Set Intensity.
Then, under the File Acquisition tab, enter a custom file name, and select a folder for saving the data files. Next, enter the number of cells to acquire. Load the first experimental sample tube and click the Acquire button.
When all of the samples have been run, open the imaging flow cytometer analysis software application, and open a single experimental RIF file. Under the compensation tab, select Create New Matrix. The software will prompt for the selection of the single color files, and merge them to create a matrix file to be selected for applying the channel compensation.
Use the Building Blocks function to set up the appropriate dot plots. Then click on the statistics function to access the cell numbers in each gate. In this representative experiment, blood samples were drawn as indicated and the concentration of NK cells per milliliter of whole blood was measured for each runner, and on average for each time point.
Three out of four runners presented a similar slight increase in NK cell count after exercise. Immediately followed by a sharp decrease, before slowly returning to normal, 24 to 48 hours after the end of the event. On average the NK cell count decreased 1.5 hours after exercise, but returned to near normal levels after 24 hours.
After calculating the cell count, the cytotoxic activity of the NK cells was immediately analyzed as just demonstrated, with an average decrease in NK cell cytotoxicity immediately, and 1.5 hours after the termination of the exercise event, that increased significantly by 24 hours post exercise. The NK cytotoxicity remained high compared to the pre-exercise levels, albeit nonsignificantly. Once mastered, this technique can be completed in about four hours when performed properly.
After watching this video, you should have a good understanding of how to obtain and characterize NK cells count and cytotoxic activity from human blood samples in a reproducible hydropic manner.
